This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this research is to use x-ray absorption spectroscopy to analyze the reaction mechanism of nitrile hydratases (NHases). The importance of this project is underscored by the extensive industrial use of nitriles to produce a broad number of specialty chemicals containing amines, amides, amidines, carboxylic acids, esters, aldehydes, ketones, and heterocyclic compounds. These compounds are used in a wide array of chemical reactions as feedstock for the production of solvents, extractants, pharmaceuticals, pesticides, and polymers. The proposed research aims to define the reaction mechanisms for both the Co-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Comamonas testosteroni (CtNHase). The successful completion of the proposed work will not only provide insight into the catalytic mechanism of NHase enzymes, but will also provide new insight into the use of NHase enzymes in organic chemical processing, industrial syntheses, and bioremediation tools.